The synthesis of nucleic acids and peptides on a solid phase has become an established process during the last 20 years. The most prevalent method of nucleic acid synthesis is the phosphoramidite method of Beaucage, S. L. and Caruthers, M. H. (Tetrahedron Lett. 22 (1981) 1859-1862), where the oligonucleotide chain is built up by the repetitive condensation of individual nucleotide building blocks in the 3′ or 5′ direction. A variety of orthogonal protecting groups are used to protect three reactive nucleotide groups: the ribose sugar 5′ hydroxyl group, the amino group of the nucleobase adenine, guanine and cytosine (thymine does not need a protecting group), as well as the phosphate group of the nucleotide 3′ phosphate residue. The 4,4′-dimethoxytriphenylmethyl (DMT) group has become the standard protecting group for 5′ hydroxyl, the 2-cyanoethyl protecting group the standard for the phosphate residue and various acyl groups the standard for the amino functions of the nucleobases according to Buchi, H. and Khorana, H. G. (J. Mol. Biol. 72 (1972), 251-288) and Souveaux (in: Methods in Molecular Biology, Vol. 26, Chap. 1 Protocol for Oligonucleoside Conjugates, S. Agrawal (ed.), Human Press Inc., Totowa, N.J. (1994)). These protecting groups are then cleaved under varying conditions, either during or after synthesis. The DMT group is cleaved off during synthesis in order to generate an hydroxyl group to which the next phosphoramidite can bind. The other named protecting groups remain until the end of the synthesis in order to prevent any side-reactions or by-products. At the end of the synthesis the complete oligonucleotide is deprotected by means of a base treatment, whereby the 2-cyanoethyl and the acyl protecting groups are cleaved.
There are essentially two ways of producing biochips, namely the off-chip and the on-chip synthesis of oligonucleotide probes. For off-chip synthesis, the oligonucleotide is produced on a commercially available synthesizer using the above-mentioned standard reagents and then immobilized on the chip. For on-chip synthesis, the oligonucleotide is produced directly on the chip using the above-mentioned standard reagents as well. In off-chip synthesis, the quality of the oligonucleotide can be analyzed by means of analytical processes such as HPLC or mass spectrometry and, where necessary, the quality can be improved via purification. In case of on-chip synthesis, only a limited quality control is applicable, whereas purification is not possible at all. Quality control for on-chip synthesis is in general only possible by means of the covalent binding of a (mainly fluorescent) label at the terminus of the oligonucleotide, which can then be detected and quantified.
The state of the art describes already the manufacturing of arrays with spatial addressable electrochemical on-chip synthesis. CIS BIO International describes a method to synthesize an array by using an electronically conductive copolymer and an electrochemical coupling procedure (EP 0 691 978). The arrays consists of a device with one or more electrodes, whereas the surface of the electrodes are coated with said conductive copolymer. Therefore, the conductive copolymer and the synthesized polymers are directly attached to the electrodes. After synthesis, the conductive copolymer together with the attached nucleotide polymers is used for hybridization reactions and the detection thereof.
Montgomery describes a synthesis method for the preparation of polymers using electrochemical placement of monomers at specific locations on a substrate containing at least one electrode (WO 98/01221). The substrate provides at its surface at least one electrode that is proximate to at least one molecule bearing at least one protected chemical functional group. Montgomery also describes the electrochemical solid phase synthesis for the preparation of diverse sequences of separate polymers or nucleic acids at a specific location on a substrate in the U.S. Pat. No. 6,093,302. The patent claims a method for the electrochemical placement of a material at a specific location on a substrate having at its surface at least one electrode that is proximate to at least one molecule, wherein said molecule is either directly attached to the surface of said substrate or it is attached to the surface of said substrate via a linker molecule or it is attached to a layer of material overlaying said substrate.
Another attempt to synthesize large numbers of polymers is disclosed by Southern (WO 93/22480). Southern describes a method for synthesizing polymers at selected sites by electrochemically modifying a surface, whereas this method comprises an electrolyte overlaying the surface and an array of electrodes adjacent to said surface. In each step of the synthesis process, an array of electrodes is mechanically placed adjacent to the surface in order to modify the surface. The array of electrodes is then mechanically removed and the surface is subsequently contacted with selected monomers. For subsequent reactions, the array of electrodes is again mechanically placed adjacent to the surface. This method requires a large amount of control to position the electrodes repeatedly on the surface, providing the necessary accuracy.